Abstract Blood transfusions remain an important treatment modality for patients with sickle cell disease (SCD) and thalassemia (Thal). However, 15-50% of these patients develop alloantibodies causing complications ranging from difficulty in finding matched units to potentially life-threatening delayed hemolytic transfusion reactions (DHTR). Increasing evidence suggest that free hemoglobin (Hb) and heme play central roles in many aspects of the pathophysiology of hemolytic anemias, especially in SCD. With regards to alloimmunization, we have previously identified weakened innate immunoregulatory function in response to free heme in alloimmunized SCD patients with the implication that hemolysis is a potential alloimmunization risk factor. Our preliminary data indicate that free heme can directly inhibit B plasma B cell differentiation in healthy donors and non- alloimmunized but not alloimmunized SCD patients. Heme/hemolysis resulted in enhancement of T follicular regulatory cell (TFR) suppressive activity, key in inhibiting B cell responses, in non-alloimmunized but not alloimmunized SCD patients. We therefore posit that defective response to heme/hemolysis in B cells and TFR cell increases their risk of alloimmunization. We hypothesize that heme-sensing pathways in key humoral immune cells are critical in the regulation of SCD RBC alloimmunization. We will test our hypothesis by examining key DOCK8/HO-1/ROS heme molecular pathways at basal level and during RBC transfusion in B cells (Aim1) and TFR cells (Aim2) in patients with SCD. Using both in vitro and in vivo models, we will also test the efficacy of drugs that target these pathways to switch off humoral immune response against transfused RBCs under hemolytic conditions. We believe that elucidation of molecular pathways associated with the defective heme response in humoral immune cells in alloimmunized patients is likely to lead to identification of potential biomarkers of alloimmunization and DHTR in hemolytic disorders and novel therapeutic targets that can prevent or even inhibit alloimmunization/DHTR.